Several publications are referenced in this application to describe the state of the art to which the invention pertains. Each of these publications is incorporated by reference herein.
The aryl hydrocarbon receptor (AH receptor or AHR), AH receptor nuclear transporter (ARNT), Drosophila single-minded gene product (SIM) and Drosophila period gene product (PER) are the founding members of an emerging superfamily of regulatory proteins. The AHR and ARNT are heterodimeric partners that transcriptionally upregulate genes involved in the metabolism of xenobiotics. The AHR is activatable by a number of widespread environmental pollutants like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In the absence of agonist, the AHR is primarily cytosolic and functionally repressed, presumably as the result of its tight association with Hsp90. Current models suggest that agonist binding initiates translocation of the receptor complex to the nucleus and concomitantly weakens the AHR-Hsp90 association. Within the nucleus, Hsp90 is displaced and the AHR dimerizes with its partner ARNT resulting in a bHLH-PAS heterodimer with binding specificity for DNA sequences within enhancer elements upstream of gene products that metabolize foreign chemicals. In Drosophila, SIM is master regulator of midline cell lineage in the embryonic nervous system. In vitro and in vivo studies suggest that SIM may also dimerize with an ARNT-like protein to regulate enhancer sequences present in the sim, slit and Toll structural genes. The Drosophila PER protein plays a role in the maintenance of circadian rhythms. PER has been shown to form heterotypic interactions with a second Drosophila protein, TIM, in vivo, and homotypic interactions with the ARNT molecule in vitro.
The distinguishing characteristic of these proteins is a 200-300 stretch of amino acid sequence similarity known as a PAS (PER/ARNT/SIM) domain. In the AHR, the PAS domain has been shown to encode sites for agonist binding, surfaces to support heterodimerization with other PAS domains, as well as surfaces that form tight interactions with Hsp90. In addition to the PAS domain, the AHR, ARNT and SIM also harbor a bHLH (basic helix-loop-helix) motif that plays a primary role in dimer formation. The bHLH motif is found in a variety of transcription factors that utilize homotypic interactions to regulate various aspects of cell growth and differentiation. Dimerization specificity is dictated by sequences within both the bHLH and determinants within secondary interaction surfaces, such as the “leucine zipper or PAS domains. Interestingly, these dimerization surfaces also appear to restrict pairing to within a given bHLH protein superfamily, thus minimizing crosstalk between important cellular pathways.
Because other bHLH protein families utilize multiple homotypic interactions to provide fine control in the regulation of certain gene batteries, it is possible that additional bHLH-PAS proteins exist in the mammalian genome and that a subset of these proteins might dimerize with either the AHR or ARNT. However, prior to the present invention, the AHR and ARNT were the only mammalian bHLH-PAS proteins that had been identified. Accordingly, a need exists to identify and characterize other bHLH-PAS domain proteins, particularly those that are novel receptors for drugs, or are AHR or ARNT binding partners. Such molecules would find broad utility as research tools in elucidating environmentally and developmentally controlled signal transduction pathways, and also as diagnostic and therapeutic agents for detection and control of such pathways.